Orientation Determination of a Hybrid Peptide Immobilized on CVD

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Orientation Determination of a Hybrid Peptide Immobilized on CVDBased Reactive Polymer Surfaces Shuai Wei,† Xingquan Zou,† Kenneth Cheng,§ Joshua Jasensky,† Qiuming Wang,† Yaoxin Li,† Christoph Hussal,⊥ Joerg Lahann,§ Charles L. Brooks III,*,†,‡ and Zhan Chen*,†,‡ †

Department of Chemistry, ‡Department of Biophysics, and §Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States ⊥ Institute of Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldsshafen, Germany ABSTRACT: Antimicrobial peptides (AMPs), due to their unique structure/function relationship, have great opportunities to be developed into novel diagnostic and therapeutic agents for a variety of pathogens and illnesses. Often such peptides are administered using powder or suspension, limiting their reusability or recyclability. Immobilization of these antimicrobial peptides on biotic/abiotic surfaces may circumvent such disadvantages, but such immobilization is likely to not only change the peptide secondary structures and their orientations but also ultimately affect functionality. In order to better understand surface-bound structures of AMPs on abiotic surfaces, cecropin A (1−8)−melittin (1−18) hybrid peptides were chemically immobilized on polymer surfaces prepared by chemical vapor deposition (CVD) polymerization. Measurements by sum frequency generation (SFG) vibrational spectroscopy and circular dichroism were used to characterize the peptides immobilized on the CVD-based polymer in situ. In addition, coarse-grained molecular dynamics (MD) simulations were used to understand the orientation of these peptides on the molecular level. Simulation results were highly consistent with experimental data. Results indicated that, unlike other linear peptides immobilized on similar abiotic surfaces, this hybrid peptide immobilized on CVD-based polymer surfaces exhibited two bending points. Such conclusions help further understand the role surface immobilization for such unique molecules.

1. INTRODUCTION In an effort to combat bacterial drug resistance, the antimicrobial peptide (AMP) has been extensively investigated for its potential use as an alternative to conventional antibiotic therapy.1−4 AMPs are regarded as a key component of the natural immunity of an organism against bacteria or harmful biological substance; its antibiotic mechanism involves permeation of the bacterial membrane to the point of inducing cell lysis and eventually killing the bacteria.1−3 Several models have been proposed in order to explain the membrane permeability of AMPs; primary factors that influence such interactions include AMP’s amino acid composition, amphipathicity, and cationic charge.1,5 The 37-residue cecropin A is one of the most-well-studied AMPs; it exhibits broad-spectrum activity against bacteria, low activity against eukaryotic cells, and virtually no hemolytic activity.6,7 Melittin is another wellstudied nonspecific 26-residue peptide toxin that exhibits strong antimicrobial and hemolytic activity.8,9 Significant work has been done to improve the potency and specificity of AMPs against pathogenic agents while their cytotoxic effect toward eukaryotic cells is minimized. Researchers discovered that AMP sequence hybridization10,11 from cecropin A and melittin results in a better-functioning AMP by both improving the © XXXX American Chemical Society

antimicrobial activity relative to cecropin A and greatly reducing its hemolytic properties relative to melittin.12 Cecropin A (1−8)−melittin (1−18) hybrid peptide is one of the most successful examples of this hybrid conception and has been extensively studied in terms of antimicrobial activity, as well as membrane interaction behaviors.13−16 The majority of studies about this cecropin A (1−8)− melittin (1−18) hybrid peptide have been performed in bulk solution.10,11 Surface tethering is an attractive and alternative approach because it promotes the reusability of such biomolecules. For surface-immobilized hybrid peptides, understanding their structure (such as conformation and orientation) after surface immobilization is quite important, because such structure must have a great impact on peptide functionality. Immobilized peptides may have a tendency to adopt a particular conformation (e.g., α-helix) and orientation with surface normal, which can be observed by using sum frequency generation (SFG) vibrational spectroscopy.17−21 SFG has been extensively used to study amino acids, peptides, and proteins at Received: March 16, 2016 Revised: August 4, 2016

A

DOI: 10.1021/acs.jpcc.6b02751 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C interfaces,22−37 including the studies on the orientation determination of surface-immobilized peptides, many of which adopt α-helix structure.17,19,38−40 In addition to SFG experiments, the detailed linear helical structure and the specific peptide orientation of surface-immobilized peptides have also been captured and validated in several studies using a coarsegrained molecular dynamics (MD) method.41 MD results can provide more details about peptide structures after surface immobilization. Such residue-level detailed structure and orientation obtained from MD simulations can lead to an indepth understanding on peptide-surface interactions and also potentially provide a guide to design a peptide to adopt a preferred conformation (e.g., α-helix) and a desired orientation on a surface. Such MD simulation predictions can in turn be validated by SFG experimental studies. In fact, not all peptides immobilized on surfaces which adopt α-helical structures possess an ideal linear helical structure without disruption. Melittin, with a proline located at residue number 22 (P22), is expected to have a helical structure with a kink around position P22, as proline itself does not readily form hydrogen bonds in a helix.42 Chen et al.43 have investigated the orientations of melittin associated with a solid-supported single lipid bilayer. In order to accommodate the bend structure of melittin, they represented melittin as being composed of two independent α-helices with a fixed angle between the two segments. Ding et al.44 studied such nonlinear α-helical structures of free peptide associated with lipid bilayers. In this work, the orientation information on a nonlinear α-helix on a lipid bilayer could be obtained by using either a bent structure or a disrupted structure. It was defined that a bent structure has a direction change in the helix with all the residues remaining helical, while the disrupted structure not only has a direction change of the helix axis but the amino acid(s) at the change also lose their helical characteristics, which means that the phase of the amide I E vibrational mode of the helix is not continuous at the kink position.44 With this knowledge in mind, our goal is to investigate cecropin−melittin hybrid peptides chemically immobilized to a solid support. In our previous investigations, solid support surfaces for peptide immobilization are usually based on selfassembled monolayers (SAMs).20,21,40 SAMs present an ideal model for such studies because they are highly ordered and simple. However, SAMs may not be able to form with such a high quality on many substrates, which greatly limits their practical applications. In this research, we studied peptide immobilization on a polymer surface created by chemical vapor deposition (CVD) polymerization. Such a polymer surface can be deposited on any substrates and can have a much wider range of applications. This research effort used a combination of three techniquesSFG, coarse-grained MD simulation, and circular dichroism (CD) spectroscopyall working in tandem to determine the orientation and structure of the surfaceimmobilized hybrid peptide. Compared to other linear helical peptides, the innate bent structure of the cecropin A (1−8)− melittin (1−18) hybrid peptide is more complicated, which needs further development in the preexisting models to deduce peptide orientation. Results of this study provide a successful case that will potentially guide the analysis of surfaceimmobilized helical AMPs with complicated structures.

a cysteine residue at the C-terminus was synthesized by Peptide 2.0 Inc. (Chantilly, VA). Right-angle CaF2 prisms were purchased from Altos Photonics (Bozeman, MT). All chemicals such as sodium dodecyl sulfate (SDS), tris(2-carboxyethyl)phosphine (TCEP), and phosphate buffer (PB) were purchased from Sigma-Aldrich (Saint Louis, MO) and used as received without further purification. 2.2. CVD Polymer. The synthesis of the precursor, 4-(3,4dibromomaleimide)[2.2]paracyclophane, has been described elsewhere.45 CVD polymerization was performed using a custom-built CVD system previously developed.46 All substrates were fixed inside the CVD deposition chamber at 13 °C. The precursor underwent sublimation (∼120 °C) and pyrolysis (750 °C) to form a stream of reactive diradical vapor. Subsequently, the diradical vapor entered the CVD deposition chamber, where it deposited and polymerized on the substrates to form a thin layer of polymer with dibromomaleimide pendant groups. The whole process operated at a reduced pressure of 0.1 mbar with the use of argon as the carrier gas. A schematic showing the molecular formula of the polymer substrate is displayed in Figure 1.

Figure 1. Molecular formula of the dibromomaleimide-functionalized CVD polymer.

2.3. SFG Measurement and Orientation Determination. The SFG spectroscopy is a second-order nonlinear optical technique that has submonolayer surface and interface sensitivity. Details regarding SFG theory and applications have been extensively published47−51 and will not be repeated here. The setup used in this study was a commercial SFG system from EKSPLA. The laser delivers pulses of 20 ps at a repetition rate of 50 Hz. The generated SFG intensity is normalized by the intensities of the visible and infrared beams and then plotted as a function of infrared frequency. SFG spectra are collected with different polarization combinations of the input visible, input IR, and generated SFG beams, including ssp (s-polarized SFG signal, s-polarized visible input, and ppolarized input IR) and ppp. The SFG signal intensities detected using different polarization combinations of the laser beams in near total reflection geometry are primarily contributed by their corresponding second-order nonlinear (2) optical susceptibility tensor components, e.g., χ(2) zzz and χyyz . By (2) (2) taking the ratio of χzzz and χyyz measured from different polarization combinations, the factor of peptide density N can be eliminated. The SFG signal intensity can be expressed as

ISFG ∝ |χ (2) |2 I visIIR

(1)

and χ(2) can be written as48 (2) χ (2) = χNR +

2. EXPERIMENTAL SECTION 2.1. Materials. The hybrid peptide (sequence KWKLFKKIGIGAVLKVLTTGLPALISC, MW = 2898 g/mol, ≥98%) with

∑ q

Aq ωIR − ωq + iΓq

(2)

χ(2) NR

where is the nonresonant contribution, ωIR is the frequency of the IR beam, and Aq, ωq, and Γq are the strength, resonant B

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Figure 2. (a) The near-total-reflection geometry of the SFG experiment. (b) Time-dependent monitoring of peptide immobilization via SFG (at 1650 cm−1).

frequency, and damping constant of the qth vibrational mode, respectively.49 The SFG spectra are normalized by the intensities of visible (Ivis) and IR (IIR) beams to eliminate the effect of laser intensity fluctuations. The SFG spectra of ppp and ssp polarization combinations are fitted using |χ(2)|2 to obtain quantitative vibrational strength and then to calculate (2) the χ(2) zzz/χyyz ratio, which is used to deduce peptide orientation. In order to obtain orientation information, we need to connect the second-order susceptibility χ(2) of a vibrational model of peptides measured in the lab coordinate system with the hyperpolarizability β(2) of the mode in the molecular coordinate system. For an α-helical structure, χ(2) of the amide I mode is a function of helix tilt angle θ and hyperpolarizability β(2), where the tilt angle θ represents the angle between the principal axis of the α-helix peptide and the surface normal and hyperpolarizability β(2) can be obtained from the product of the Raman polarizability derivative and the infrared dipole moment derivative of an amide I mode. More details on SFG orientation analysis on α-helical structure can be found in previous publications.43,52,53 2.4. Surface Immobilization of Peptides. The hybrid peptide immobilization process was monitored in a timedependent SFG measurement at 1650 cm−1. A CVD-coated CaF2 prism was mounted on top of (and in contact with) a phosphate buffer (pH 7.2) solution containing a low concentration of TCEP (5 nM). A near-total-reflection geometry was used for SFG signal collection, where the 532 nm green beam and infrared beam go through one side of the right angle CaF2 prism and then overlap spatially and temporary on the other side, as shown in Figure 2a. The hybrid peptide solution (86 μM, 100 μL) was added to the 2 mL phosphate buffer solution at time zero. A magnetic stir bar was used at a rate of 75 rpm to ensure a homogeneous concentration distribution of the peptides added to the PB solution in contact with the prism. TCEP reduces the disulfide bonds formed between peptide cysteine residues and promotes covalent bond formation between the polymer surface dibromomaleimide groups and peptides. As seen in Figure 2b, the SFG signal starts to increase, showing that peptide molecules are immobilized onto the surface. The signal increase reaches equilibrium at 3.3 h. To ensure that all the peptides on the CVD polymer surface are chemically immobilized, we washed the polymer surface (with peptides) with 1% SDS detergent and PB solution to remove the physically adsorbed

hybrid peptides (if any). SFG spectra were then collected from the surface-immobilized peptides in contact with phosphate buffer using ppp and ssp polarization combinations. 2.5. Circular Dichroism (CD) Spectra Measurement. The secondary structure of the hybrid peptides immobilized on the CVD polymer was measured by a J-1500 CD spectrometer (JASCO Inc.) using a continuous scanning mode at room temperature. High-quality z-cut quartz slides were used as substrates for CVD polymer coating and hybrid peptide immobilization. After removing physically adsorbed hybrid peptides with a 1% sodium dodecyl sulfate (SDS) and phosphate buffer (PB) solution wash, the CD spectrum was collected between 240 and 190 nm at a 1 nm resolution and 50 nm/min scan rate, and five scans were averaged. The CD signal was hardly detectable when only one quartz slide with immobilized peptides was used. To increase the CD signal, 10 slides with immobilized peptides were stacked together in the buffer solution and CD spectra were collected.

3. SIMULATION METHODS 3.1. Models for Peptide and Surface. A perfect α-helical structure was used as the initial template for the hybrid peptide with an extra cysteine residue on the C-terminus. Each initial structure of the hybrid is relaxed with energy minimization using CHARMM54 in implicit solvent. The relaxed structure was then submitted to the Go model builder on the MMTSB Web site (http://www.mmtsb.org) to generate an input file for coarse-grained simulations. Such a hybrid peptide is shown in Figure 3 and is color-coded to show its sequence origins: melittin (1−18) (orange) and cecropin A (1−8) (cyan). The proline residue (P22) close to the C-terminus of the peptide has a very high helix propensity index of 3.16 (very unlikely to be involved in an ideal helical structure). Therefore, the helical structure is bent at the position of the proline residue. 3.2. Simulation Model. The protein model used in this work is the Karanicolas and Brooks (KB) Go-like model.55,56 This model is a one-bead coarse-grained model with defined native contacts to determine protein folding. The model has been shown in many cases to be successful in reproducing protein folding free energy surfaces and folding mechanisms.57−61 The KB model has also been successfully applied to studies on protein−peptide interactions with repulsive surface models.62−65 A coarse-grained potential for the protein−surface interactions was recently developed on the C

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time step of 10 fs. As discussed in previous studies of the surface force field parametrization and application to a similar peptide interacting with a different SAM surface, the simulation temperature is set to be 215 K to represent the experimental temperature of 298 K. A different temperature (215 K) was used in the simulation study from room temperature (298 K) because the temperature of the coarse-grained MD simulation method could not be directly correlated to the real experimental temperature. At 215 K, most proteins form a folded structure simulated with the coarse-grained model, which have been shown to reproduce the protein adsorption free energies and stabilities on surfaces, similar to the results obtained using experiments performed at 298 K.40,41,66 Therefore, the simulation temperature of 215 K has been widely used in previous MD simulations as well as in this work. Also, as shown in a previous simulation study of a different peptide, the thermal stability of cecropin P1 (CP1), an AMP with α-helical secondary structure, is highly improved by tethering to the SAM surface by either terminal residue (over 100 K higher than in the solution).66 Therefore, we initiate the simulation by assuming a helical structure on the surface at the simulation temperature. The angle formed by the helical peptide (assuming a single helix is formed) and the surface normal is one order parameter to measure in the simulation, which is comparable to the SFG experiments. The orientation of the helix will be calculated as

Figure 3. A cartoon representation of the hybrid peptide as the initial structure for simulations.

basis of and to be utilized with the KB Go-like protein model,40,41,66 which is shown as ⎧ ⎡ ⎛ ⎞9 ⎛ σi ⎞7 ⎛ σi ⎞3 ⎪ σi 3 ⎢ ⎨ = ∑ πρσi ϵi θ1⎜ ⎟ − θ2⎜ ⎟ + θ3⎜ ⎟ ⎪ ⎢⎣ ⎝ zis ⎠ ⎝ zi s ⎠ ⎝ zi s ⎠ i ⎩ N

Vsurface

⎛ σi ⎞3⎤⎫ ⎪ − (θs(χs − 4.5) + θpχp )⎜ ⎟ ⎥⎬ ⎥ ⎝ zi s ⎠ ⎦ ⎪ ⎭

(3)

where N is the number of residues in the peptide/protein, zis is the distance between residue i and the surface, and σi and ϵi are residue-specific van der Waals parameters. The parameters (shown in Table 1) used in this work were determined in the previous study.41

⎛ z − z0 ⎞ ⎟ ξ = a cos⎜ ⎝ r ⎠

where z is the surface normal coordinate of the untethered terminus, z0 is the z-coordinate of the residue that is adsorbed on the surface closest to the untethered terminus, and r is the distance between these two residues. As will be shown, this hybrid peptide may form a bent helical structure on the surface. In this case, as shown in Figure 4, we

Table 1. Parameters for the Surface Model41 θ1

θ2

θ3

θs

θp

0.2340

0.4936

0.1333

0.0067

0.0333

(5)

As shown in eq 3, the first three terms of the potential function between the protein and the surface successfully capture the adsorption well and the energy barrier features as observed in experimental studies.64,65 Furthermore, the twothird power terms were added to the function to account for hydrophobic effects of different SAM surfaces and different residues in a protein or peptide by using the hydrophobic index of the surface (χs) and amino acid (χp).58 For the dibromomaleimide-functionalized CVD polymer surface, we use a value of χs = 4.5 to account for its hydrophobic property. The tethering bond from the terminal cysteine residue to the dibromomaleimide group is modeled by a harmonic restraint with an interaction potential of the form 1 Urestraint = k r(r − req)2 (4) 2 where kr = 10 kcal/mol is the parameter describing the strength of the restraint and r is the distance of the restrained site from its original position of (0,0,0) Å. The equilibrium length is req = 5.8 Å, which approximately represents the distance between the surface and Cα of the cysteine residue at the tethering site. 3.3. Molecular Dynamics (MD) Simulation. In order to explore hybrid peptide orientation on the dibromomaleimidefunctionalized CVD polymer surface, MD simulations were performed within the canonical ensemble (NVT) at a specific temperature. Each simulation was performed with 10 million steps of equilibrium and 100 million steps of production with a

Figure 4. Cartoon representation of the definition of angles ξ and ς.

measured both the angle ξ formed by helix I (the one close to the surface) and the surface normal and the angle ς between helix I and helix II (the one further away from the surface). The (2) χ(2) ppp/χssp ratio as a function of these angles can be calculated by considering a disrupted α-helical structure with two segments. We can sum the contributions of the Raman polarizability and the IR transition dipole moment from each segment to calculate the SFG hyperpolarizability tensor of a disrupted helix. (2) The χ(2) ppp/χssp ratio at any particular orientation angle can then be obtained with the assumption of a single orientation (δdistribution) of all the surface-immobilized peptides. A detailed orientation analysis of helical structures can be found in previous publications.38,43,49,67 Using the values of ξ and ς D

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The Journal of Physical Chemistry C (2) deduced through simulation, the distribution of χ(2) ppp/χssp ratios can be predicted. The percentage of the helical motif in this small peptide is measured from simulation using a novel secondary structure assignment method, PCASSO, developed by Law et al.68 This method is fast, efficient, and specific to Cα-only structures, which is an ideal method to analyze structures from the KB Golike model.

hybrid peptide immobilized on a CVD polymer surface in phosphate buffer. A typical CD spectrum collected from an αhelical structure exhibits two negative bands at 222 and 208 nm and a positive peak at 190 nm.69 The CD spectrum from the immobilized hybrid peptides on the CVD polymer clearly shows such characteristics, indicating that such hybrid peptides mainly adopt an α-helical structure. We determined that the helix content is 48.4% in the immobilized hybrid peptide by fitting the spectrum using the DichroWeb server (http:// dichroweb.cryst.bbk.ac.uk/).70 To quantify the secondary structure formation through MD simulations, a recently developed method, PCASSO, was used to assign secondary structures. The fraction of helical formation for each residue is plotted in Figure 6a. As shown, the helical strand is broken into three noncontinuous parts. In Figure 6b, a snapshot of a typical structure obtained from the same simulation shows the detailed helical structure orientations. From the N-terminus, the first helical strand spans residues 2− 6 (five-residue-long “helix II”), which is far away from the surface and shown in cyan. The middle subhelix is the longest and is formed by residues 12−19 (eight-residue-long “helix I”), which is only a few residues away from the surface. Another short structure created by residues 22−24 (three residues long) is observed and assigned to be helical; however, we believe that it is too short to contribute either CD signal or SFG signal. Nevertheless, both the CD experiment and the simulation show consistency; the hybrid peptide on the CVD polymer surface has substantial helical content. It is interesting to notice that there are two kinks found in this structure. The first kink is at a position close to the surface as a result of the residue P22, which is unlikely to form a helical structure. The second kink happens at the hybrid point of the two helices from different origins. This result indicates that this human-designed hybrid point is not stable enough to keep the peptide helical structure when the peptide is tethered on the surface. Therefore, it suggests that a further development of peptide hybridization is needed if a peptide is desired to keep a single helical structure (which is beyond the scope of this research). 4.2. Orientation Study of Hybrid Peptides Immobilized on CVD Polymer. Orientation determination is very important in applications of surface-immobilized hybrid peptides, since peptide orientation influences the properties of immobilized peptides. It is also able to provide molecular

4. RESULTS AND DISCUSSION 4.1. Secondary Structure of Hybrid Peptides Immobilized on CVD Polymer. The secondary structure of the hybrid peptide cecropin A (1−8)−melittin (1−18) in solution has been investigated in previous studies.16 The peptide predominately exhibits a β-sheet in aqueous buffer solution but adopts an α-helical structure in the presence of a lipid membrane. In order to investigate the secondary structure in chemically immobilized hybrid peptide molecules on surfaces, we used a combination of CD spectroscopy and coarse-grained molecular dynamics simulation to investigate the secondary structure of hybrid peptides immobilized on a solid polymer surface. Figure 5 displays a representative CD spectrum of

Figure 5. CD spectrum of hybrid peptides immobilized on the CVD polymer in phosphate buffer.

Figure 6. Helical structure formation for each residue in the hybrid peptide and a snapshot of a typical peptide structure on the CVD polymer surface. (a) The PCASSO-estimated helical structure formation. (b) A snapshot of a typical structure of the peptide on CVD polymer from the simulation. E

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Figure 7. SFG spectra collected in the amide I region at ppp and ssp polarization combinations. (a) Background spectra of the CVD polymer/ phosphate buffer interface before hybrid peptide immobilization. (b) SFG spectra of immobilized hybrid peptides on CVD polymer surface in contact with phosphate buffer.

(2) Figure 8. (a) Helix orientation angle distributions with ξ (blue) and ς (red) and (b) χ(2) ppp/χssp density distribution of surface-immobilized hybrid peptide.

cecropin A (1−8)−melittin (1−18) hybrid peptide studied here also contains this proline from melittin. Here the hybridization between cecropin A and melittin also disrupts the helical structure and forms a bend, which was confirmed by simulation. Compared with linear cecropin A, it is challenging to determine the orientation of hybrid peptides immobilized on the polymer surface due to these structural deviations from an ideal helical structure. The two kinks in the cecropin A (1−8)− melittin (1−18) hybrid peptide change the SFG hyperpolarizability β(2), which in turn, changes the relationship (2) (2) between the χppp /χssp ratio and the orientation angle. Therefore, to determine the orientation of the breaking helical strands immobilized on the polymer surface, the simulation trajectory was analyzed to obtain the orientation angle distributions of immobilized peptides. As shown in Figure 8a, the distribution of the tilt angle formed by helix I and the surface normal is plotted as the blue curve, and the red curve indicates the distribution of the bending angle ς that is formed between the two helices. The average tilt angle is around 61° and the averaged bending angle (2) is 35°. The χ(2) ppp/χssp ratio from SFG is then calculated according to previously developed theoretical methods18,43,44,49,67 using the length of helices and the orientations of each segment from the simulation trajectory. The density (2) distribution of the calculated χ(2) ppp/χssp is then plotted in Figure

insight into how hybrid peptides interact with the CVD polymer surface. Before immobilization of hybrid peptides, the SFG signal of CVD polymer in contact with phosphate buffer solution was measured in the amide I region between 1500 and 1800 cm−1, as shown in Figure 7a. A small peak located at 1720 cm−1 observed in the ppp spectrum originates from the CO functional groups on the CVD polymer surface. After peptide immobilization, a strong amide I peak centered at 1650 cm−1 was detected in both ppp and ssp spectra (Figure 7b). This 1650 cm−1 amide I peak is generated from the α-helical structure of the immobilized hybrid peptide, which confirms the results obtained from CD study and coarse-grained molecular dynamics simulation. After we fit the SFG ssp and ppp spectra collected from the immobilized hybrid peptide shown in Figure 7b, we found that (2) the measured χ(2) ppp/χssp ratio is 1.23. This value is not located (2) within the range (1.6−2.8) of the χ(2) ppp/χssp ratio of a δdistribution of orientation by assuming a perfect linear helical structure. We believe that this deviation of the experimentally measured result from the calculated value range of a linear helical structure is due to the bending structure of the immobilized hybrid peptides, which was obtained from MD simulations. Previous studies71,72 show that the proline amino acid in melittin gives rise to a flexible hinge region and results in a significant deviation of melittin from an ideal linear helix. A F

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The Journal of Physical Chemistry C (2) 8b. The χ(2) ppp/χssp distribution curve shows a high peak around 1.2 that is consistent with SFG experimental measurements (measured to be a ratio of 1.23). Also, as discussed, we believe that the short lying down helical strand around the C-terminus does not contribute to the SFG signal and is ignored in the (2) calculation of the χ(2) ppp/χssp ratio. It is worth noting that the orientation distribution of the hybrid peptide on the CVD polymer surface is broad, which suggests that the peptide has good rotational flexibility as tethered to the surface. The good flexibility is expected mainly due to the P22 at the first kink region. This study clearly demonstrates that the incorporation between experimental and simulation methods provides detailed structural and orientation knowledge for a complicated peptide conformation on the surface. CD data provides conformation information on surface-immobilized peptides, while SFG measures both conformation (through peak centers) and orientation (through SFG signals detected using different polarization combinations). The MD simulation data can provide more details on the structure of the surfaceimmobilized peptides, and the simulation results can be validated using SFG in a quantitative fashion.

study a nonlinear and disrupted helical peptide immobilized on a surface. It is important to elucidate structure−function relationships of immobilized peptides on polymer surfaces for future applications.

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AUTHOR INFORMATION

Corresponding Authors

*C.L.B.: e-mail, [email protected]; phone, (+1)734-6476682. *Z.C.: e-mail, [email protected]; phone, (+1)734-615-6628. Author Contributions

S.W. and X.Z. contributed equally to this paper. The manuscript was written through contributions of all the authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Army Research Office (W911NF-11-1-0251).

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5. CONCLUSIONS The main results of this research can be summarized into the following two aspects: (1) hybrid peptide cecropin A (1−8)− melittin (1−18) has been successfully immobilized onto a CVD polymer through chemical linkage between the peptide cysteine residues and the dibromomaleimide groups on the polymer surface. Previously, we have extensively studied surfaceimmobilized peptides on SAM surfaces. SAM surfaces certainly provide excellent models to immobilize biological molecules, but their practical applications are limited. Here the CVDdeposited polymers are more widely applicable and the surface functionalities on such surfaces can be more versatile.45,73,74 Therefore, the successful immobilization of peptides on CVD polymer surfaces should find more practical applications in the future. (2) The detailed structure of the surface hybrid peptides immobilized onto a CVD polymer was successfully deduced using a combined experimental and simulation study. The CD spectrum shows that immobilized hybrid peptides adopt an αhelical structure, which was confirmed by coarse-grained MD simulation. SFG signal also indicates that the main structure of the surface-immobilized peptide is α-helix. In order to deduce the orientation of this immobilized hybrid peptide, polarized SFG spectra were collected. However, the experimentally (2) measured χ(2) ppp/χssp ratio of 1.23 does not match any calculated tilt angle of a linear α-helical peptide assuming a delta orientation angle distribution. MD simulations show that surface-immobilized hybrid peptides exhibit a bending α-helical structure with bend points at two different locations along the peptide backbone caused by peptide hybridization and a proline residue; this deviates this peptide from an ideal linear helix. A coarse-grained molecular dynamics simulation gives us an (2) average χ(2) ppp/χssp ratio of 1.2, which is consistent with the SFG measured value. Furthermore, these simulation results also indicate that for the two kinks in an immobilized peptide molecule, one is near the hybridization region and the other one is near the proline residue. In this study, the experimental studies using CD, SFG, and MD simulations showed compatible results that the surface-immobilized hybrid peptide adopted a “complicated” structure. We believe that this is the first time that these orthogonal techniques were combined to

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